Characterisation of a common hotspot variant in acute intermittent porphyria sheds light on the mechanism of hydroxymethylbilane synthase function

Hydroxymethylbilane synthase (HMBS) is the third enzyme involved in haem biosynthesis, in which it catalyses the formation of tetrapyrrole 1‐hydroxymethylbilane (HMB). In this process, HMBS binds four consecutive substrate molecules, creating the enzyme‐intermediate complexes ES, ES(2), ES(3) and ES(4). Pathogenic variants in the HMBS gene are associated with the dominantly inherited disorder acute intermittent porphyria. In this study, we have characterised the p.R26H variant to shed light on the role of Arg26 in the elongation mechanism of HMBS and to provide insights into its effect on the enzyme. With selected biophysical methods, we have been able to show that p.R26H forms a single enzyme‐intermediate complex in the ES(2)‐state. We were also able to demonstrate that the p.R26H variant results in an inactive enzyme, which is unable to produce the HMB product.